| Malignant tumor is one of the most common diseases and the major causes of mortality. The initiation, promotion and progression of malignant tumor were considered to include a series of stages from normality, precancerous lesions, carcinoma in situ to invasion and metastasis of malignant tumor. The process involves many changes at molecular level such as activation of oncogenes and inactivation of tumor suppressor genes. These changes can lead to the overproliferation, abnormal differentiation and malignant transformation of normal cells.Hepatocyte growth factor/Scatter factor (HGF/SF) is a mesenchyme-derived pleiotropic growth factor. Extracellular activation of single chain HGF is a limiting step in the c-Met signaling pathway, which is involved in various aspects of tumor biology in vivo, such as growth, differentiation, invasion, and tumor angiogenesis. The process is believed to be regulated by a serine proteinase designated as HGF activator, which is secreted mainly by the liver and circulated as an inactive zymogen. The zymogen may be activated locally by thrombin in injured tissues. HGFA activates scHGF/SF efficiently. Hepatocyte growth factor activator inhibitor(HAI-l), is a novel Kunitz-type serine protease inhibitor with efficient inhibitory activity against HGFA, while the other serum proteinase inhibitors are not responsible for inhibiting the activity of HGFA, synthesis of HA1-1 may play an important role in the local regulation of HGF/SF activity.This study focuses on the expression of HAI-1 in various tissues theimportant regulating factor of the HGF/c-Met signaling pathway at the protein level. We successfully subcloned HAI-1 cDNA fragments into expression vectors and the GST-HAI-1 fusion proteins were obtained. Monoclonal antibody against HAI-1 was prepared after immunization of BALB/C mice with recovered GST-HAI-1 fusion proteins by hybridoma technique. Distribution of HAI-1 was detected by immunohistochemistry in human tissues utilizing McAb against HAI-1. Expression of HAI-1 was analyzed to speculate the potential roles of HAI-1 in the initiation and progression of colorectal cancer.1. Expression of HAI-1 in E.coli and recovery of the fusion protein: To obtain enough amount of HAI-1 protein for further studies, the human HAI-1 cDNA fragments were subcloned into expression vectors pGEX-5X-2 and pGEX-5X-l. Restriction enzyme digestion analysis showed that pGEX-5X-2-HAI-1/EcoR I +BamH I and pGEX-5X-l-HAI-l/EcoR I + Xho I recombinant plasmids were constructed correctly. SDS-PAGE indicated that GST-HAI-1 fusion proteins were expressed in E.coli as expected. The fusion proteins were recovered by preparative SDS-PAGE and subsequent eletroelution.2. Preparation and characterization of monoclonal antibody against HAI-1: Purified HAI-1-GST fusion proteins were used to immunize BALB/C mice. Monoclonal antibody ZMC6 against HAI-1 was successfully obtained with hybridoma technique. The liters of antibodies in the ascites of BLAB/C mice determined by ELISA were 1:262,144. Western Blot confirmed that this antibody (ZMC6) could specifically recognize HAI-1 proteins expressed in the GST-HAI-1 fusion protein production E.coli and the protein extracts of human colorectal tissues.3. Distribution of HAI-1 protein in human tissues: Expression of HAI-1 in various human tissues was examined by immunohistochemistry. HAI-1 protein is mainly present in epithelial tissues, with varied amounts in normal and carcinoma tissues of colon, duodenum, stomach, breast, lung, esophagus,meninges, thyroid gland, endometrium, etc. 1. Expression of HAI-1 protein in human colorectal carcinoma:Statistical analysis showed that expression levels of HAI-1 in colorectal cancer, adjacent normal epithelium covering mucosal surface and intestinal glandular epithelium were significantly different in 38 colorectal cancer patients (Friedman nonparametric test, P<0.001). In 52 colorectal cancers, HAI-1 expression showed a significant correlati...
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